The habenula is a paired nucleus residing in the dorsal thalamus. It consists of medial and lateral subnuclei (MHb, LHb), which connect to the brainstem via a common tract, yet innervate different brainstem targets. In the initial three years of this project, we have used transgenic mice to define the gene expression patterns and output pathways of the MHb and LHb. Recent work has shown that some LHb neurons inhibit dopamine (DA) signaling in the ventral tegmental area (VTA) via local GABAergic interneurons. This pathway is thought to mediate reward prediction error and punishment, signals which could be overactive in depression, and a few preliminary human studies have linked the habenula to mechanisms of depression. Although LHb regulation of DA activity is clearly important, we find that >90% of LHb outputs project instead to the midbrain/pontine raphe nuclei and dorsal tegmental (DTg) area, including regions rich in serotonergic (5HT) neurons. Little is known about the specific connections or the behavioral function of the pathway from the LHb and the raphe/DTg. Given the known roles of 5HT in the regulation of sleep, mood, and stress, the LHb-raphe pathway may be of great clinical significance. Thus in addition to understanding LHb regulation of DA activity, additional models of LHb function are needed. In years 4-8 of this project, we will use optogenetically assisted circuit mapping to define functional pathways from the LHb to specific neurons in the raphe and DTg. We will also test the function of these LHb pathways in behavioral models of locomotion, anxiety, reinforcement and stress. Aim 1: Determine if the LHb makes direct synaptic connections with brainstem GABAergic neurons. We hypothesize that the LHb synapses with GABAergic neurons in the raphe and DTg, as it does in the VTA. We will use optogenetically-assisted circuit mapping, with Cre recombinase-driven expression of ChR2 in specific neurons, to assess the connection between the LHb and genetically-labeled GABAergic neurons in the raphe and DTg, using intracellular visual patch recording in brain slice preparations. Aim 2: Determine the direct and polysynaptic connections of the LHb with the 5HT system. In this aim we will use optogenetic circuit mapping to determine if there is a monosynaptic connection between the LHb and 5HT neurons in the raphe nuclei receiving LHb inputs. We will also use electrophysiology and inhibitors of specific neurotransmitter systems to determine if there is a polysynaptic link between the LHb and 5HT neurons in the raphe via GABAergic interneurons, analogous to the LHb-RMTg-VTA system. Aim 3: Examine the behavioral role of LHb inputs to the raphe system. Here we will express the light-activated ion channel ChR2 in the LHb, then stimulate LHb fiber terminals in the raphe nuclei in freely moving mice to determine the role of the LHb-raphe pathway in locomotion, models of anxiety, self- stimulation reinforcement, and behavioral responses to stress.